Our overall hypothesis is that RNA interference (RNAi) can be developed to reduce specific gene expression over long-term without toxic effects in human lymphocytes. RNAi using siRNA to inhibit specific gene expression is a powerful and promising technology for both basic research and therapeutic intervention. However, our results and several recent studies have shown unintended nonspecific cytotoxic effects associated with this technology in mammalian cells raising a concern for the long term use of the RNAi in therapeutic applications in humans. The cytotoxic effects were attributed at least in part to apoptosis, although other mechanisms are also possible. The cytotoxicity was associated with higher levels of shRNA expression. Based upon these results, we hypothesized that that the levels of shRNA are important for determining whether a given shRNA will be cytotoxic or not and that optimization of those levels can yield shRNAs that are both effective and not or minimally cytotoxic. We will also address whether the cell localization of the shRNA and/or its pathway for processing can affect the extent of cytotoxicity within cells. Through our proposed studies we will understand the effects of shRNA expression in mature lymphoid cells in vitro as well as the effects on the differentiation of the lymphocyte populations in an in vivo setting. We will minimize the cytotoxic effect and maximize stable and effective function of RNAi in primary T-lymphocytes. The relevance of this research to public health. RNA interference is a powerful and promising technology to inhibit specific gene expression for both basic research and therapeutic intervention. The inhibition of specific gene expression needs to be effective without unintended side effects. This proposal propose to study the effects of RNAi in human T lymphocytes to maximize the RNAi effect and minimize side effects.